The neuronal channel NALCN contributes resting sodium permeability and is required for normal respiratory rhythm

Sodium plays a key role in determining the basal excitability of the nervous systems through the resting "leak" Na(+) permeabilities, but the molecular identities of the TTX- and Cs(+)-resistant Na(+) leak conductance are totally unknown. Here we show that this conductance is formed by the protein NALCN, a substantially uncharacterized member of the sodium/calcium channel family. Unlike any of the other 20 family members, NALCN forms a voltage-independent, nonselective cation channel. NALCN mutant mice have a severely disrupted respiratory rhythm and die within 24 hours of birth. Brain stem-spinal cord recordings reveal reduced neuronal firing. The TTX- and Cs(+)-resistant background Na(+) leak current is absent in the mutant hippocampal neurons. The resting membrane potentials of the mutant neurons are relatively insensitive to changes in extracellular Na(+) concentration. Thus, NALCN, a nonselective cation channel, forms the background Na(+) leak conductance and controls neuronal excitability.     

A sodium leak current regulates pacemaker activity of adult central pattern generator neurons in Lymnaea stagnalis

The resting membrane potential of the pacemaker neurons is one of the essential mechanisms underlying rhythm generation. In this study, we described the biophysical properties of an uncharacterized channel (U-type channel) and investigated the role of the channel in the rhythmic activity of a respiratory pacemaker neuron and the respiratory behaviour in adult freshwater snail Lymnaea stagnalis. Our results show that the channel conducts an inward leak current carried by Na(+) (I(Leak-Na)). The I(Leak-Na) contributed to the resting membrane potential and was required for maintaining rhythmic action potential bursting activity of the identified pacemaker RPeD1 neurons. Partial knockdown of the U-type channel suppressed the aerial respiratory behaviour of the adult snail in vivo. These findings identified the Na(+) leak conductance via the U-type channel, likely a NALCN-like channel, as one of the fundamental mechanisms regulating rhythm activity of pacemaker neurons and respiratory behaviour in adult animals.     

Sodium leak channels in neuronal excitability and rhythmic behaviors

Extracellular K?, Na?, and Ca2? ions all influence the resting membrane potential of the neuron. However, the mechanisms by which extracellular Na? and Ca2? regulate basal neuronal excitability are not well understood. Recent findings suggest that NALCN, in association with UNC79 and UNC80, contributes a basal Na? leak conductance in neurons. Mutations in Nalcn, Unc79, or Unc80 lead to severe phenotypes that include neonatal lethality and disruption in rhythmic behaviors. This review discusses the properties of the NALCN complex, its regulation, and its contribution to neuronal function and animal behavior.     

Separable gating mechanisms in a Mammalian pacemaker channel

Despite permeability to both K(+) and Na(+), hyperpolarization-activated cyclic nucleotide-gated (HCN) pacemaker channels contain the K(+) channel signature sequence, GYG, within the selectivity filter of the pore. Here, we show that this region is involved in regulating gating in a mouse isoform of the pacemaker channel (mHCN2). A mutation in the GYG sequence of the selectivity filter (G404S) had different effects on the two components of the wild-type current; it eliminated the slowly activating current (I(f)) but, surprisingly, did not affect the instantaneous current (I(inst)). Confocal imaging and immunocytochemistry showed G404S protein on the periphery of the cells, consistent with the presence of channels on the plasma membrane. Experiments with the wild-type channel showed that the rate of I(f) deactivation and I(f) amplitude had a parallel dependence on the ratio of K(+)/Na(+) driving forces. In addition, the amplitude of fully activated I(f), unlike I(inst), was not well predicted by equal and independent flow of K(+) and Na(+). The data are consistent with two separable gating mechanisms associated with pacemaker channels: one (I(f)) that is sensitive to voltage, to a mutation in the selectivity filter, and to driving forces for permeating cations and another (I(inst)) that is insensitive to these influences.     

Effect of [Cl(-)]i on ENaC activity from mouse cortical collecting duct cells

Na(+) transport via epithelial Na(+) channel (ENaC) occurs across many epithelial surfaces and plays a key role in regulating salt and water absorption. In this study, we have examined the effects of cytosolic Na(+) and Cl(-) on ENaC activity by patch clamping single channel recording method in mouse cortical collecting duct cells (M1). Cytosolic Na(+) exerts its effect in change of ENaC open probability (Po). High cytosolic Na(+) significantly reduces ENaC Po. No change in channel conductance by cytosolic Na(+) is observed. However, decrease of cytosolic Cl(-) concentration significantly increases channel conductance and ENaC Po. This effect is due to the right shift of ENaC I-V curve to positive membrane potential. The virtue of ENaC conductance remains the same. Cl(-) channels like CFTR and VRAC are unlikely to be involved in this regulation. The results suggest that cytosolic Cl(-) could serve as a mediator to regulate ENaC activity, in accordance with the activities of Cl(-) channels.     

Essential requirement of cytosolic phospholipase A(2) for activation of the H(+) channel in phagocyte-like cells.

The NADPH oxidase-producing superoxide is the major mechanism by which phagocytes kill invading pathogens. We previously established a model of cytosolic phospholipase A(2) (cPLA(2))-deficient differentiated PLB-985 cells (PLB-D cells) and demonstrated that cPLA(2)-generated arachidonic acid (AA) is essential for NADPH oxidase activation (Dana, R., Leto, T., Malech, H., and Levy, R. (1998) J. Biol. Chem. 273, 441-445). In the present study, we used this model to determine the physiological role of cPLA(2) in the regulation of both the H(+) channel and the Na(+)/H(+) antiporter and to study whether NADPH oxidase activation is regulated by either of these transporters. PLB-D cells and two controls: parent PLB-985 cells and PLB-985 cells transfected with the vector only (PLB cells) were differentiated using 1.25% Me(2)SO or 5 x 10(-8) M 1, 25-dihydroxyvitamin D(3). Activation of differentiated PLB cells resulted in a Zn(2+)-sensitive alkalization, indicating H(+) channel activity. In contrast, differentiated PLB-D cells failed to activate the H(+) channel, but the addition of exogenous AA fully restored this activity, indicating the role of cPLA(2) in H(+) channel activation. The presence of the H(+) channel inhibitor Zn(2+) caused significant inhibition of NADPH oxidase activity, suggesting a role of the H(+) channel in regulating oxidase activity. Na(+)/H(+) antiporter activity was stimulated in differentiated PLB-D cells, indicating that cPLA(2) does not participate in the regulation of this antiporter. These results establish an essential and specific physiological requirement of cPLA(2)-generated AA for activation of the H(+) channel and suggest the participation of this channel in the regulation of NADPH oxidase activity.     

Na(+) current contribution to the diastolic depolarization in newborn rabbit SA node cells

Isolated newborn, but not adult, rabbit sinoatrial node (SAN) cells exhibit spontaneous activity that (unlike adult) are highly sensitive to the Na(+) current (I(Na)) blocker TTX. To investigate this TTX action on automaticity, cells were voltage clamped with ramp depolarizations mimicking the pacemaker phase of spontaneous cells (-60 to -20 mV, 35 mV/s). Ramps elicited a TTX-sensitive current in newborn (peak density 0.89 +/- 0.14 pA/pF, n = 24) but not adult (n = 5) cells. When depolarizing ramps were preceded by steplike depolarizations to mimic action potentials, ramp current decreased 54.6 +/- 8.0% (n = 3) but was not abolished. Additional experiments demonstrated that ramp current amplitude depended on the slope of the ramp and that TTX did not alter steady-state holding current at pacemaker potentials. This excluded a steady-state Na(+) window component and suggested a kinetic basis, which was investigated by measuring TTX-sensitive I(Na) during long step depolarizations. I(Na) exhibited a slow but complete inactivation time course at pacemaker voltages (tau = 33.9 +/- 3.9 ms at -50 mV), consistent with the rate-dependent ramp data. The data indicate that owing to slow inactivation of I(Na) at diastolic potentials, a small TTX-sensitive current flows during the diastolic depolarization in neonatal pacemaker myocytes.     

The NALCN ion channel is activated by M3 muscarinic receptors in a pancreatic β-cell line

A previously uncharacterized putative ion channel, NALCN (sodium leak channel, non-selective), has been recently shown to be responsible for the tetrodotoxin (TTX)-resistant sodium leak current implicated in the regulation of neuronal excitability. Here, we show that NALCN encodes a current that is activated by M3 muscarinic receptors (M3R) in a pancreatic β-cell line. This current is primarily permeant to sodium ions, independent of intracellular calcium stores and G proteins but dependent on Src activation, and resistant to TTX. The current is recapitulated by co-expression of NALCN and M3R in human embryonic kidney-293 cells and in Xenopus oocytes. We also show that NALCN and M3R belong to the same protein complex, involving the intracellular I–II loop of NALCN and the intracellular i3 loop of M3R. Taken together, our data show the molecular basis of a muscarinic-activated inward sodium current that is independent of G-protein activation, and provide new insights into the properties of NALCN channels.     

A uniquely adaptable pore is consistent with NALCN being an ion sensor

NALCN is an intriguing, orphan ion channel among the 4x6TM family of related voltage-gated cation channels, sharing a common architecture of four homologous domains consisting of six transmembrane helices, separated by three cytoplasmic linkers and delimited by N and C-terminal ends. NALCN is one of the shortest 4x6TM family members, lacking much of the variation that provides the diverse palate of gating features, and tissue specific adaptations of sodium and calcium channels. NALCN’s most distinctive feature is that that it possesses a highly adaptable pore with a calcium-like EEEE selectivity filter in radially symmetrical animals and a more sodium-like EEKE or EKEE selectivity filter in bilaterally symmetrical animals including vertebrates. Two lineages of animals evolved alternative calcium-like EEEE and sodium-like EEKE / EKEE pores, spliced to regulate NALCN functions in differing cellular environments, such as muscle (heart and skeletal) and secretory tissue (brain and glands), respectively. A highly adaptable pore in an otherwise conserved ion channel in the 4x6TM channel family is not consistent with a role for NALCN in directly gating a significant ion conductance that can be either sodium ions or calcium ions. NALCN was proposed to be an expressible Gd³⁺-sensitive, NMDG⁺-impermeant, non-selective and ohmic leak conductance in HEK-293T cells, but we were unable to distinguish these reported currents from leaky patch currents (I_LP) in control HEK-293T cells. We suggest that NALCN functions as a sensor for the much larger UNC80/UNC79 complex, in a manner consistent with the coupling mechanism known for other weakly or non-conducting 4x6TM channel sensor proteins such as Na_x or Ca_v1.1. We propose that NALCN serves as a variable sensor that responds to calcium or sodium ion flux, depending on whether the total cellular current density is generated more from calcium-selective or sodium-selective channels.     

A mutation in telethonin alters Nav1.5 function

Excitable cells express a variety of ion channels that allow rapid exchange of ions with the extracellular space. Opening of Na(+) channels in excitable cells results in influx of Na(+) and cellular depolarization. The function of Na(v)1.5, an Na(+) channel expressed in the heart, brain, and gastrointestinal tract, is altered by interacting proteins. The pore-forming alpha-subunit of this channel is encoded by SCN5A. Genetic perturbations in SCN5A cause type 3 long QT syndrome and type 1 Brugada syndrome, two distinct heritable arrhythmia syndromes. Mutations in SCN5A are also associated with increased prevalence of gastrointestinal symptoms, suggesting that the Na(+) channel plays a role in normal gastrointestinal physiology and that alterations in its function may cause disease. We collected blood from patients with intestinal pseudo-obstruction (a disease associated with abnormal motility in the gut) and screened for mutations in SCN5A and ion channel-interacting proteins. A 42-year-old male patient was found to have a mutation in the gene TCAP, encoding for the small protein telethonin. Telethonin was found to be expressed in the human gastrointestinal smooth muscle, co-localized with Na(v)1.5, and co-immunoprecipitated with sodium channels. Expression of mutated telethonin, when co-expressed with SCN5A in HEK 293 cells, altered steady state activation kinetics of SCN5A, resulting in a doubling of the window current. These results suggest a new role for telethonin, namely that telethonin is a sodium channel-interacting protein. Also, mutations in telethonin can alter Na(v)1.5 kinetics and may play a role in intestinal pseudo-obstruction.     

Pacemaker activity and ionic currents in mouse atrioventricular node cells

It is well established that Pacemaker activity of the sino-atrial node (SAN) initiates the heartbeat. However, the atrioventricular node (AVN) can generate viable pacemaker activity in case of SAN failure, but we have limited knowledge of the ionic bases of AVN automaticity. We characterized pacemaker activity and ionic currents in automatic myocytes of the mouse AVN. Pacemaking of AVN cells (AVNCs) was lower than that of SAN pacemaker cells (SANCs), both in control conditions and upon perfusion of isoproterenol (ISO). Block of I(Na) by tetrodotoxin (TTX) or of I(Ca,L) by isradipine abolished AVNCs pacemaker activity. TTX-resistant (I(Nar)) and TTX-sensitive (I(Nas)) Na(+) currents were recorded in mouse AVNCs, as well as T-(I(Ca,T)) and L-type (I(Ca,L)) Ca(2+) currents I(Ca,L) density was lower than in SANCs (51%). The density of the hyperpolarization-activated current, (I(f)) and that of the fast component of the delayed rectifier current (I(Kr)) were, respectively, lower (52%) and higher (53%) in AVNCs than in SANCs. Pharmacological inhibition of I(f) by 3 μM ZD-7228 reduced pacemaker activity by 16%, suggesting a relevant role for I(f) in AVNCs automaticity. Some AVNCs expressed also moderate densities of the transient outward K(+) current (I(to)). In contrast, no detectable slow component of the delayed rectifier current (I(Ks)) could be recorded in AVNCs. The lower densities of I(f) and I(Ca,L), as well as higher expression of I(Kr) in AVNCs than in SANCs may contribute to the intrinsically slower AVNCs pacemaking than that of SANCs.     

Y1767C, a novel SCN5A mutation, induces a persistent Na+ current and potentiates ranolazine inhibition of Nav1.5 channels

Long QT syndrome type 3 (LQT3) has been traced to mutations of the cardiac Na(+) channel (Na(v)1.5) that produce persistent Na(+) currents leading to delayed ventricular repolarization and torsades de pointes. We performed mutational analyses of patients suffering from LQTS and characterized the biophysical properties of the mutations that we uncovered. One LQT3 patient carried a mutation in the SCN5A gene in which the cysteine was substituted for a highly conserved tyrosine (Y1767C) located near the cytoplasmic entrance of the Na(v)1.5 channel pore. The wild-type and mutant channels were transiently expressed in tsA201 cells, and Na(+) currents were recorded using the patch-clamp technique. The Y1767C channel produced a persistent Na(+) current, more rapid inactivation, faster recovery from inactivation, and an increased window current. The persistent Na(+) current of the Y1767C channel was blocked by ranolazine but not by many class I antiarrhythmic drugs. The incomplete inactivation, along with the persistent activation of Na(+) channels caused by an overlap of voltage-dependent activation and inactivation, known as window currents, appeared to contribute to the LQTS phenotype in this patient. The blocking effect of ranolazine on the persistent Na(+) current suggested that ranolazine may be an effective therapeutic treatment for patients with this mutation. Our data also revealed the unique role for the Y1767 residue in inactivating and forming the intracellular pore of the Na(v)1.5 channel.     

A weakly voltage-dependent, nonselective cation channel mediates toxic sodium influx in wheat

To determine the transporters responsible for toxic Na(+) influx in wheat (Triticum aestivum), root plasma membrane preparations were screened using the planar lipid bilayer technique as an assay for Na(+)-permeable ion channel activity. The predominant channel in the bilayer was a 44-pS channel that we called the nonselective cation (NSC) channel, which was nonselective for monovalent cations and weakly voltage dependent. Single channel characteristics of the NSC channel were compared with (22)Na(+) influx into excised root segments. Na(+) influx through the NSC channel resembled (22)Na(+) influx in its partial sensitivity to inhibition by Ca(2+), Mg(2+), and Gd(3+), and its insensitivity to all other inhibitors tested (tetraethylammonium, quinine, Cs(+), tetrodotoxin, verapamil, amiloride, and flufenamate). Na(+) influx through the NSC channel also closely resembled an instantaneous current in wheat root protoplasts (S.D. Tyerman, M. Skerrett, A. Garill, G.P. Findlay, R. Leigh [1997] J Exp Bot 48: 459-480) in its permeability sequence, selectivity for K(+) over Na(+) (approximately 1.25), insensitivity to tetraethylammonium, voltage independence, and partial sensitivity to Ca(2+). Comparison of tissue, protoplast (S.D. Tyerman, M. Skerrett, A. Garill, G.P. Findlay, R. Leigh [1997] J Exp Bot 48: 459-480), and single- channel data indicate that toxic Na(+) influx is catalyzed by a single transporter, and this is likely to be the NSC channel identified in planar lipid bilayers.     

Contribution of Nav1.1 to cellular synchronization and automaticity in spontaneous beating cultured neonatal rat ventricular cells

Two factors of cell coupling influence cellular synchronization and automaticity: gap junction coupling and ion channels activity. However, the role of Na(+) channel isoforms underlying cell-to-cell interaction and cellular automaticity is not well understood. To address these questions, we studied mRNA expression of Na(+) channel isoforms and the effects of TTX on spontaneously beating cultured ventricle cells. Using RT-PCR technique we demonstrated the presence of Na(v)1.1 and Na(v)1.5 channels. The reduction of Na(v)1.1 channel activity disturbed cell-to-cell interaction and changed beating rates. Thus, Na(v)1.1 channel is involved in cellular synchronization and automaticity.     

Molecular identification of a TTX-sensitive Ca(2+) current

The TTX-sensitive Ca(2+) current [I(Ca(TTX))] observed in cardiac myocytes under Na(+)-free conditions was investigated using patch-clamp and Ca(2+)-imaging methods. Cs(+) and Ca(2+) were found to contribute to I(Ca(TTX)), but TEA(+) and N-methyl-D-glucamine (NMDG(+)) did not. HEK-293 cells transfected with cardiac Na(+) channels exhibited a current that resembled I(Ca(TTX)) in cardiac myocytes with regard to voltage dependence, inactivation kinetics, and ion selectivity, suggesting that the cardiac Na(+) channel itself gives rise to I(Ca(TTX)). Furthermore, repeated activation of I(Ca(TTX)) led to a 60% increase in intracellular Ca(2+) concentration, confirming Ca(2+) entry through this current. Ba(2+) permeation of I(Ca(TTX)), reported by others, did not occur in rat myocytes or in HEK-293 cells expressing cardiac Na(+) channels under our experimental conditions. The report of block of I(Ca(TTX)) in guinea pig heart by mibefradil (10 microM) was supported in transfected HEK-293 cells, but Na(+) current was also blocked (half-block at 0.45 microM). We conclude that I(Ca(TTX)) reflects current through cardiac Na(+) channels in Na(+)-free (or "null") conditions. We suggest that the current be renamed I(Na(null)) to more accurately reflect the molecular identity of the channel and the conditions needed for its activation. The relationship between I(Na(null)) and Ca(2+) flux through slip-mode conductance of cardiac Na(+) channels is discussed in the context of ion channel biophysics and "permeation plasticity."     

Extracellular calcium controls background current and neuronal excitability via an UNC79-UNC80-NALCN cation channel complex

In contrast to its extensively studied intracellular roles, the molecular mechanisms by which extracellular Ca(2+) regulates the basal excitability of neurons are unclear. One mechanism is believed to be through Ca(2+)'s interaction with the negative charges on the cell membrane (the charge screening effect). Here we show that, in cultured hippocampal neurons, lowering [Ca(2+)](e) activates a NALCN channel-dependent Na(+)-leak current (I(L-Na)). The coupling between [Ca(2+)](e) and NALCN requires a Ca(2+)-sensing G protein-coupled receptor, an activation of G-proteins, an UNC80 protein that bridges NALCN to a large novel protein UNC79 in the same complex, and the last amino acid of NALCN's intracellular tail. In neurons from nalcn and unc79 knockout mice, I(L-Na) is insensitive to changes in [Ca(2+)](e), and reducing [Ca(2+)](e) fails to elicit the excitatory effects seen in the wild-type. Therefore, extracellular Ca(2+) influences neuronal excitability through the UNC79-UNC80-NALCN complex in a G protein-dependent fashion.